Project Summary The long-term goal of this application is to optimize the design of novel, non-toxic inhibitors of myeloperoxidase (MPO) to inhibit atherosclerosis. MPO is highly expressed in inflammatory phagocytes and is considered to play an important role in host defense mechanisms. However, phagocytes are also activated by oxidized biomolecules that are found in the vessel wall. Upon activation these inflammatory phagocytes release MPO and begin to generate hydrogen peroxide (H2O2), which is required to activate MPO. Once activated, MPO generates a wide variety of potent oxidants and secondary radicals. For example, activated MPO reacts with chloride (Cl-) to generate hypochlorous acid (HOCl). MPO catalytically consumes nitric oxide (NO) and converts it to nitrite (NO2-). In turn, MPO oxidizes NO2- to generate nitrogen dioxide (NO2), a radical that is capable of oxidizing lipids, proteins and DNA. MPO also oxidizes aromatic amino acids such as tyrosine and tryptophan to generate cytotoxic tyrosyl and tryptophanyl radicals. As MPO generates such a wide variety of oxidants and radicals that impair endothelial-dependent vasodilatation and accelerate atherosclerosis, it is imperative that effective, non-toxic inhibitors of MPO be developed. In this application, we propose to design and develop novel tripeptide competitive inhibitors of MPO. In Aim 1, systematic amino acid substitutions will be used to optimize inhibitor design. Effects of the inhibitors on MPO activity will be determined in in vitro systems using both purified MPO and macrophages isolated from MPO-/- and transgenic human MPO (Tg-h- MPO+/+) mice. Further, effects of the inhibitors on cellular cytotoxicity will be determined with respect to macrophage cell number, cytokine production and macrophage foam cell formation. Aim 2 has two major goals. First, Aim 2 will determine the pharmacokinetics and cytotoxicity of the most effective MPO inhibitors identified in Aim 1. These studies will be performed in C57BL/6J mice. Mice will be injected intraperitoneally (ip) and plasma levels of inhibitor will be determined by HPLC with respect to dose and time. Plasma AST and ALT levels will be used to monitor liver function while histology will be used to monitor the effects of the tripeptides on cell structure of the major organs. Second, Aim 2 will determine whether the tripeptide inhibitors from Aim 1 will improve vasodilatation and inhibit atherosclerosis in chimeric Ldlr-/-/MPO-/- and Ldlr-/- Tg-h-MPO+/+ mice fed western diet. Endothelium-dependent vasodilatation will be determined by videomicroscopy. Atherosclerotic lesion formation will be determined histologically using immunofluorescence and confocal microscopy. Successful completion of the proposed studies will result in the development of a new class of non-toxic MPO inhibitors that improve vasodilatation and prevent atherosclerosis. The optimized MPO inhibitors developed here should also be useful for treating vascular disease and inflammation in other disease states such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, inflammatory bowel disease, kidney disease, rheumatoid arthritis and chronic obstructive pulmonary disease, where aberrant MPO activity has been implicated. As these disease states represent a major focus of the NIH, findings from our proposal will advance the research and mission of several different institutes.